Projects started in fiscal year 2009 according to goals (A to D) listed under 'Goals and Objectives': (A1) Development of a hamster model for Ebola virus infection We have established a uniformly lethal model in Syrian golden hamsters using mouse-adapted Ebola virus (EBOV). Hamsters showed severe coagulation abnormalities resembling disseminated intravascular coagulation as seen in patients. Pathological investigation suggested that severe hepatocellular damage and massive cellular depletions in lymphoid organs were the main causes of death. Cytokine gene profiles showed suppression of inflammatory cytokine expression early after infection and massive cytokine up-regulation (cytokine storm) at terminal stages of disease, which both seem to contribute to pathogenesis. (A2) Development of a guinea pig model for Marburg virus infection The Angolan strain of Marburg virus (MARV) is significantly more virulent than other MARV strains. We have established a lethal guinea pig by three sequential passages of the Angola strain in this animal species. The guinea pig-adapted MARV Angola strain is uniformly lethal and causes death within 10 days. Genome sequencing identified a single amino acid change in the matrix protein VP40 as the virulence factor. (A3) Development of a hamster model for New World hantaviruses. To better understand the pathogenesis of hantavirus pulmonary syndrome (HPS) we have established a Andes virus animal model in Syrian golden hamster. In order to study the pathogenicity of other HPS causing viruses, we have initiated experiments aimed at adapting Sin Nombre virus to hamsters. The objective of these studies is to perform comparative genetic analysis on the virus during serial passage to identify determinants of virulence acquisition. (A4) Severe Acute Respiratory Syndrome (SARS) in Cynomolgus macaques. Cynomolgus macaques were infected with 3 different SARS-coronavirus mutants. None of the animals showed overt clinical signs but radiological differences were observed between the 3 viruses. Samples were processed for virus titrations, RNA extraction and histology. Plasma samples from each clinical exam were tested for levels of 23 cytokines. Virus titers were determined for all tissues. We are in the progress of titering the organs from this study and awaiting histopathology. (B1) Studying host responses to infection in Syrian golden hamster disease models Syrian golden hamsters have been broadly used in infectious disease research. However, in contrast to mouse and nonhuman primate models appropriate tools to detect host responses are very limited. Therefore, we first developed quantitative real time RT-PCR for monitoring 60 hamster immune response genes. Subsequently, we started a project to sequence the transcriptome of the Syrian golden hamster. All sequence data are back and we are currently in the process of annotation for 35,000 genes. The final goal of this project is the development of an expression microarray chip that can be utilized for pathogenesis and other studies. (B2) Immune responses to hantavirus infections It is thought that the pathogenic potential of hantaviruses correlates with a mal-adaptive immune response to infection. We are interested in the interaction between the virus and the innate and adaptive immune response using both in vitro and in vivo model systems. We have recently discovered that cells defective in type I interferon are able to respond to infection by secreting interferon lambda, a newly recognized cytokine which acts as a link between the innate and adaptive immune response. As of yet, the pattern recognition receptor used by human endothelial cells is undetermined. Currently we are developing assays to introduce short hairpin RNAs and dominant negative constructs into primary human endothelial cells directed towards the PRR pathway to elucidate the mechanisms of innate immune activation. (B3) Antagonism of interferon (IFN) mediated innate immunity by hantaviruses. We hypothesize that hantavirus replication and establishment of persistent infection is mediated in part via evasion of interferon-directed host innate immune responses. Research, to date, has focused on investigating the putative antagonistic function of individual viral proteins. We could confirm that the glycoproteins of pathogenic New World hantaviruses appear to be the primary antagonist of RIG-I directed IFN production. However, in contrast to published literature that implicates the glycoprotein as the main antagonist in innate immunity signaling, our preliminary results suggest that the nucleocapsid protein (NP) serves as the primary antagonist in JAK-STAT signaling. Furthermore, there may be a difference in the antagonistic effect of the NP based on the pathogenicity of the species from which is it expressed. (B4) Development of a cross-protective Ebola vaccine. Currently we know five species of EBOV: Zaire, Sudan, Cote dIvoire, Reston and Bundibugyo ebolavirus. VSV vaccine vectors expressing the EBOV-GPs were generated and in vitro characterized. In mice, vaccination with the Sudan, Cote dIvoire and Reston EBOV-GP expressing VSV vectors demonstrated cross-protection against lethal infection. In contrast, guinea pigs could only be protected against lethal infection when vaccinated with a VSV vaccine expressing the homologous, but not a heterologous, EBOV-GP. In additional experiments, guinea pigs infected with wildtype EBOV species were fully protected against lethal disease independent on the source of the initial infection. The data suggest that cross-protection against multiple EBOV species is possible;however the EBOV-GP alone does not seem to be sufficient in inducing protective immunity. (C1) Rodent-borne pathogen surveillance in Mali Recently a fatal case of Lassa fever was documented in a young man who worked in Southern Mali, marking the first time this disease had been documented in that country. Lassa fever is caused by Lassa virus, a rodent borne pathogen belonging to the Arenaviridae. In collaboration with Dr. Schwans group, we went to Mali to assist in his field work, and to collect samples from the captured rodents to assess the prevalence of Lassa, and other rodent-borne pathogens in Mali. In total we collected 412 samples from 103 rodents and testing will begin within the next month. (D1) Development of Henipavirus expression plasmids and VSV pseudotype viruses. We are in the progress of cloning the Nipah and Hendravirus glycoproteins (G) and fusion (F) proteins into an expression plasmids (pCAGGS) for the production of recombinant G and F protein as well as into a VSV&#8710;G plasmid with the goal to generate a recombinant VSV expressing the G and F proteins of Nipah or Hendraviruses. (D2) The importance of cathepsin cleavage for filovirus replication. Cellular cystein proteases cathepsin B &L have been shown to play an important role in Ebola virus. The goal is to analyze cathepsin B &L cleavage for all filovirus glycoproteins (GP) as well as chimeric proteins, containing parts of Ebola virus GP, using a Vesicular-Stomatitis-Virus based pseudoparticle system and reverse genetics to generate recombinant viruses. Eventually cathepsin dependent Ebola replication will be tested in vivo using cathepsin B &L ko mice. Preliminary datas suggest that Ebola virus replication is cathepsin B but not cathepsin L dependent. Current work is focused on confirming these findings. (E) Not strated yet.